Kinases involved in the regulation of energy homeostasis

ABSTRACT

The present invention discloses Inositol hexakisphosphate kinase or RYK kinase homologous proteins regulating the energy homeostasis and the metabolism of energy storage metabolites, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of metabolic diseases and disorders.

This invention relates to the use of nucleic acid sequences encodingInositol hexakisphosphate kinase or RYK kinase or homologous proteins,and the polypeptides encoded thereby and to the use of these sequencesor effectors of Inositol hexakisphosphate kinase or RYK kinase nucleicacids or polypeptides, particularly inhibitors or activators, in thediagnosis, study, prevention, and treatment of diseases and disordersrelated to body-weight regulation, for example, but not limited to,metabolic diseases such as obesity as well as related disorders such asmetabolic syndrome, eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, and gallstones.

There are several metabolic diseases of human and animal metabolism,eg., obesity and severe weight loss, that relate to energy imbalancewhere caloric intake versus energy expenditure is imbalanced. Obesity isone of the most prevalent metabolic disorders in the world. It is stilla poorly understood human disease that becomes more and more relevantfor western society. Obesity is defined as an excess of body fat,frequently resulting in a significant impairment of health.Cardiovascular risk factors like hypertension, high blood levels oftriglycerides and fasting glucose as well as low blood levels of HDLcholesterol are often linked to obesity. This typical cluster ofsymptoms is commonly defined as “metabolic syndrome” (Reaven, 2002,Circulation 106(3): 286-8 reviewed). The metabolic syndrome oftenprecedes the development of type II diabetes and cardiovascular disease(McCook, 2002, JAMA 288:2709-2716). Human obesity is strongly influencedby environmental and genetic factors, whereby the environmentalinfluence is often a hurdle for the identification of (human) obesitygenes. Obesity is influenced by genetic, metabolic, biochemical,psychological, and behavioral factors. As such, it is a complex disorderthat must be addressed on several fronts to achieve lasting positiveclinical outcome.

The molecular factors regulating food intake and body weight balance areincompletely understood. Even if several candidate genes have beendescribed which are supposed to influence the homeostatic system(s) thatregulate body mass/weight, like leptin, VCPI, VCPL, or the peroxisomeproliferator-activated receptor-gamma co-activator, the distinctmolecular mechanisms and/or molecules influencing obesity or bodyweight/body mass regulations are not known. In addition, severalsingle-gene mutations resulting in obesity have been described in mice,implicating genetic factors in the etiology of obesity (Friedman J. M.and Leibel R. L., (1992) Cell 69(2): 217-220). In the ob mouse a singlegene mutation (obese) results in profound obesity, which is accompaniedby diabetes (Friedman J. M. et al., (1991) Genomics 11: 1054-1062).

Insulin resistance greatly increases the risk of developing themetabolic syndrome (Reaven, 2002, Circulation 106(3): 286-8 reviewed).The metabolic syndrome often precedes the development of type IIdiabetes and cardiovascular disease (McCook, 2002, JAMA 288: 2709-2716).The control of blood lipid levels and blood glucose levels is theessential for the treatment of the Metabolic Syndrome (see, for example,Santomauro A. T. et al., (1999) Diabetes, 48(9): 1836-1841). Insulinamongst other hormones plays a key role in the regulation of the fuelmetabolism. High blood glucose levels stimulate the secretion of insulinby pancreatic beta-cells. Insulin leads to the storage of glycogen andtriglycerides and to the synthesis of proteins. The entry of glucoseinto muscles and adipose cells is stimulated by insulin. In patients whosuffer from diabetes mellitus either the amount of insulin produced bythe pancreatic islet cells is to low (Diabetes Type 1 or insulindependent diabetes mellitus IDDM) or liver and muscle cells loose theirability to respond to normal blood insulin levels (insulin resistance).In the next stage pancreatic cells become unable to produce sufficientamounts of insulin (Diabetes Type II or non insulin dependent diabetesmellitus NIDDM).

Therefore, the technical problem underlying the present invention was toprovide for means and methods for modulating (pathological) metabolicconditions influencing body-weight regulation and/or energy homeostaticcircuits. The solution to said technical problem is achieved byproviding the embodiments characterized in the claims.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.The present invention discloses a specific gene involved in theregulation of body-weight, energy homeostasis, metabolism, and obesity,and thus in disorders related thereto such as metabolic syndrome, eatingdisorder, cachexia, diabetes mellitus, hypertension, coronary heartdisease, hypercholesterolemia, dyslipidemia, osteoarthritis, andgallstones. The present invention describes the human Inositolhexakisphosphate kinase (3, 1, or 2) or RYK kinase gene as beinginvolved in those conditions mentioned above.

Reversible transfer of phosphate groups to substrates as proteins,lipids or other organic compounds is the main strategy for controllingactivities of eukaryotic cells. Many known signal transduction pathwaysare cascades of phosphate donor and acceptor molecules that aremodulated in function by the state of phosphorylation. Kinases areenzymes that transfer phosphate groups to specific substrates.

Inositol (1,4,5) trisphosphate is a messenger molecule that releasescalcium from intracellular stores. Polyphosphate homologues with up toeight phosphate groups have been identified, including pyrophosphates.Inositol pyrophosphates are formed by several enzymes including Inositolhexakisphosphate kinases. Members of the inositol hexakisphosphatekinase family phosphorylate inositol hexakisphosphate todiphosphoinositol pentakisphosphate, which is a ‘high energy’ candidateof cell trafficking. ‘High-energy’ pyrophosphates may play importantroles in protein phosphorylation. In mammalians (human and mouse), threeinositol hexakisphosphate kinases are known. Inositol hexakisphosphatekinase 1 (IHKPK1) and inositol hexakisphosphate kinase 2 (IHKPK2)phosphorylate inositol hexakisphosphate (IHKP) to diphosphoinositolpentakisphosphate, a candidate regulator of cellular trafficking(Saiardi A. et al., (2000) J Biol Chem 275(32):24686-24692). IHKPK2 hasbeen described as positive regulator of apoptosis and mediates growthsuppressive and apoptotic effects of interferon-beta in ovariancarcinoma cells (Morrison B. H. et al., (2001) J Biol Chem276(27):24965-14970). IHKPK3 displays a more basic character than theother two enzymes IHKPK1 and IHKPK2. IHKPK3 is most enriched in thebrain where its localization resembles IHKPK1 and IHKPK2. Intracellulardisposition discriminates the three enzymes with IHKPK2 beingexclusively nuclear, IHKPK3 predominating in the cytoplasm, and IHKPK1displaying comparable nuclear and cytosolic densities (Saiardi A. etal., (2001) J Biol Chem 276(42):39179-39185).

The Drosophila gene doughnut on 2 (dnt) encodes for a receptor tyrosinekinase-like protein that is 70% identical to the Drosophila Derailed(RYK) protein. Dnt is expressed in invaginating cells duringembryogenesis in Drosophila (Savant-Bhonsale, S. et al., 1999, Gene231(1-2): 155-61). As shown in this invention, the Drosophila dnt kinaseis most homologous to Drosophila drl and human tyrosine-protein kinaseRYK precursor, herein referred to as RYK kinase.

RYK kinase is an atypical member of the family of growth factor receptorprotein tyrosine kinases, having different activation and nucleotidebinding domains. This kinase belongs to a subfamily whose members do notappear to be regulated by phosphorylation in the activation segment. ARYK kinase ligand has not been identified so far. The protein does notshow detectable autophosphorylation activity in vitro but is capable toactivate the MAPK pathway (Katso, R. M., 1999, MCB19 (9): 6427-40).

So far, it has not been described that Inositol hexakisphosphate kinase(3, 1, or 2) or RYK kinase protein is involved in the regulation ofenergy homeostasis and body-weight regulation and related disorders, andthus, no functions in metabolic diseases and other diseases as listedabove have been discussed. In this invention we demonstrate that thecorrect gene dose of Inositol hexakisphosphate kinase or RYK kinase isessential for maintenance of energy homeostasis. A genetic screen wasused to identify that mutation of a Inositol hexakisphosphate kinase orRYK kinase homologous gene causes obesity, reflected by a significantincrease of triglyceride content, the major energy storage substance.

Polynucleotides encoding a protein with homologies to Inositolhexakisphosphate kinase or RYK kinase are suitable to investigatediseases and disorders as described above. Further new compositionsuseful in diagnosis, treatment, and prognosis of diseases and disordersas described above are provided.

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologies,which are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure.

The present invention discloses that Inositol hexakisphosphate kinase orRYK kinase homologous proteins are regulating the energy homeostasis andfat metabolism especially the metabolism and storage of triglycerides,and polynucleotides, which identify and encode the proteins disclosed inthis invention. The invention also relates to vectors, host cells,antibodies, and recombinant methods for producing the polypeptides andpolynucleotides of the invention. The invention also relates to the useof these sequences in the diagnosis, study, prevention, and treatment ofdiseases and disorders, for example, but not limited to, metabolicdiseases such as obesity as well as related disorders such as metabolicsyndrome, eating disorder, cachexia, diabetes mellitus, hypertension,coronary heart disease, hypercholesterolemia, dyslipidemia,osteoarthritis, and gallstones.

The term “polynucleotide comprising the nucleotide sequence as shown inGenBank Accession number” relates to the expressible gene of thenucleotide sequences deposited under the corresponding GenBank Accessionnumber. The term “GenBank Accession Number” relates to NCBI GenBankdatabase entries (Ref.: Benson et al., (2000) Nucleic Acids Res. 28:15-18).

Inositol hexakisphosphate kinase or RYK kinase homologous proteins andnucleic acid molecules coding therefore are obtainable from insect orvertebrate species, e.g. mammals or birds. Particularly preferred arehomologous nucleic acids, particularly nucleic acids encoding humaninositol hexakisphosphate kinase 3, human inositol hexakisphosphatekinase 1, human inositol hexakisphosphate kinase 2, or a human Drlkinase.

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides, wherein said nucleic acid moleculecomprises

-   -   (a) the nucleotide sequence of (i) GadFly Accession Number        CG10082, or inositol hexakisphosphate kinase 3 (SEQ ID NO: 1),        inositol hexakisphosphate kinase 1 (SEQ ID NO: 3), or inositol        hexakisphosphate kinase 2 (SEQ ID NO: 5), or (ii) GadFly        Accession Number CG17559, or human Drl kinase (SEQ ID NO: 9),        and/or a sequence complementary thereto,    -   (b) a nucleotide sequence which hybridizes at 50° C. in a        solution containing 1×SSC and 0.1% SDS to a sequence of (a),    -   (c) a sequence corresponding to the sequences of (a) or (b)        within the degeneration of the genetic code,    -   (d) a sequence which encodes a polypeptide which is at least        85%, preferably at least 90%, more preferably at least 95%, more        preferably at least 98% and up to 99.6% identical to the amino        acid sequences of Inositol hexakisphosphate kinase or RYK kinase        protein, preferably inositol hexakisphosphate kinase 3 (SEQ ID        NO:2), inositol hexakisphosphate kinase 1 (SEQ ID NO: 4),        inositol hexakisphosphate kinase 2 (SEQ ID NO: 6), and/or of        human RYK kinase (SEQ ID NO:10),    -   (e) a sequence which differs from the nucleic acid molecule        of (a) to (d) by mutation and wherein said mutation causes an        alteration, deletion, duplication and/or premature stop in the        encoded polypeptide or    -   (f) a partial sequence of any of the nucleotide sequences of (a)        to (e) having a length of at least 15 bases, preferably at least        20 bases, more preferably at least 25 bases and most preferably        at least 50 bases.

The invention is based on the finding that Inositol hexakisphosphatekinase or RYK kinase homologous proteins and the polynucleotidesencoding these, are involved in the regulation of triglyceride storageand therefore energy homeostasis. The invention describes the use ofthese polypeptides or fragments thereof, polynucleotides or fragmentsthereof and effectors (receptors) of these molecules, e.g. antibodies,biologically active nucleic acids, such as antisense molecules, RNAimolecules or ribozymes, aptamers, peptides or low-molecular weightorganic compounds recognizing said polynucleotides or polypeptides forthe diagnosis, study, prevention, or treatment of diseases and disordersrelated thereto, including metabolic diseases such as obesity as well asrelated disorders such as metabolic syndrome, eating disorder, cachexia,diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, and gallstones.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.To find genes with novel functions in energy homeostasis, metabolism,and obesity, a functional genetic screen was performed with the modelorganism Drosophila melanogaster (Meigen). The ability to manipulate andscreen the genomes of model organisms such as the fly Drosophilamelanogaster provides a powerful tool to analyze biological andbiochemical processes that have direct relevance to more complexvertebrate organisms due to significant evolutionary conservation ofgenes, cellular processes, and pathways (see, for example, Adams M. D.et al., (2000) Science 287: 2185-2195). Identification of novel genefunctions in model organisms can directly contribute to the elucidationof correlative pathways in mammals (humans) and of methods of modulatingthem. A correlation between a pathology model (such as changes intriglyceride levels as indication for metabolic syncrome includingobesity) and the modified expression of a fly gene can identify theassociation of the human ortholog with the particular human disease.

In one embodiment, a forward genetic screen is performed in flydisplaying a mutant phenotype due to misexpression of a known gene (see,Johnston Nat Rev Genet 3: 176-188 (2002); Rorth P., (1996) Proc NatlAcad Sci USA 93: 12418-12422). In this invention, we have used a geneticscreen to identify gene mutations that cause canges in the body weightwhich is reflected by a significant change of triglyceride levels.Resources for screening were a Drosophila melanogaster stock collectionof EP-lines. The P-vector of this collection has Gal4-UAS-binding sitesfused to a basal promoter that can transcribe adjacent genomicDrosophila sequences upon binding of Gal4 to UAS-sites. This enables theEP-line collection for overexpression of endogenous flanking genesequences. In addition, without activation of the UAS-sites, integrationof the EP-element into the gene is likely to cause a reduction of geneactivity, and allows determining its function by evaluating theloss-of-function phenotype.

Triglycerides are the most efficient storage for energy in cells, andobese people mainly show a significant increase in the content oftriglycerides. In order to isolate genes with a function in energyhomeostasis, several thousand EP-lines were tested for theirtriglyceride content after a prolonged feeding period (see Examples formore detail). Lines with significantly changed triglyceride content wereselected as positive candidates for further analysis. Additionally,glycogen levels were analysed.

In one embodiment, male flies homozygous for the integration of vectorsfor Drosophila lines EP(2)0712 and HD-EP(2)21861, respectively, wereanalyzed in an assay measuring the triglyceride contents of these flies,illustrated in more detail in the EXAMPLES section of this invention.The results of the triglyceride content analysis are shown in FIG. 1 andFIG. 8A, respectively. The average triglyceride level of the flycollection in which the fly line was found is shown as 100% in FIG. 1and FIG. 8A (first column, EP-control). The average increase oftriglyceride content of the homozygous viable Drosophila line EP(2)0712(referred to as ‘EP(2)0712’ in this invention) is 50% (see FIG. 1,second column, ‘EP(2)0712’). The average increase of triglyceridecontent of the homozygous viable Drosophila line HD-EP(2)21861 (referredto as ‘HD-EP(2)21861’ in this invention) is 70% (see FIG. 8A, secondcolumn, ‘HD-EP(2)21861’). It was found in this invention that homozygousEP(2)0712 flies and HD-EP(2)21861 flies have a significant highertriglyceride content than the control flies tested. The increase oftriglyceride content due to the loss of a gene function suggests geneactivities in energy homeostasis in a dose dependent manner thatcontrols the amount of energy stored as triglycerides.

These results were confirmed by further findings obtained by studying aDrosophila Gadfly accession number CG17348 derailed (drl) mutant. drl isthe second RYK-ortholog in Drosophila and we found, that this mutantdisplays the same metabolic phenotype as the dnt mutant, confirming aconserved metabolic function of ryk-homolog proteins in Drosophila. Inaddition to an increased triglyceride content, the glycogen content iselevated due to the loss of a gene function (see FIG. 8B). Glycogen is alarge branched polymer of glucose residues that is mainly stored inliver and muscle cells. Glycogen synthesis and degradation is central tothe control of the blood glucose level. The results are clearlyindicating a role of drl in energy homeostasis and glucose regulation.

Nucleic acids encoding the Inositol hexakisphosphate kinase or RYKkinase proteins of the present invention were identified using aplasmid-rescue or iPCR technique. Genomic DNA sequences for the inositolhexakisphosphate kinase homologous gene were isolated that are localisedadjacent to the EP vector. Using those isolated genomic sequences publicdatabases like Berkeley Drosophila Genome Project (GadFly) were screenedthereby confirming the homozygous viable integration site of thevectors. For example, EP(2)0712 vector is integrated 5′ into the cDNA ofthe Drosophila gene in antisense orientation, identified as BerkeleyDrosophila Genome Project Accession No. CG10082 (FIG. 2). In anotherexample, HD-EP(2)21861 vector is integrated into the first large intronof doughnut on 2 (dnt) gene in antisense orientation, identified asBerkeley Drosophila Genome Project Accession No. CG17559 (FIG. 9). FIG.2 and FIG. 9 show the molecular organisation of these gene loci. Thechromosomal localization site of the integration of the vector ofEP(2)0712 is at gene locus 2R, 57F6 and of the vector of HD-EP(2)21861is at gene locus 2L, 37D4-37D6. In FIG. 2 and FIG. 9, genomic DNAsequence is represented as a black dotted line in the middle thatincludes the integration site of the vectors. Numbers represent thecoordinates of the genomic DNA. Grey bars on the two “cDNA”-linesrepresent the predicted genes (as predicted by the Berkeley DrosophilaGenome Project, GadFly and by Magpie). Predicted exons of the DrosophilacDNA are shown as dark grey bars and predicted introns as light greybars.

The Drosophila genes and proteins encoded thereby with functions in theregulation of triglyceride metabolism were further analysed in publiclyavailable sequence databases (see EXAMPLES for more detail) andmammalian homologs were identified (see FIGS. 3A and 3H and FIG. 10A).

The function of the mammalian homologs in energy homeostasis was furthervalidated in this invention by analyzing the expression of thetranscripts in different tissues and by analyzing the role in adipocytedifferentiation. Expression profiling studies (see Examples for moredetail) confirm the particular relevance of the protein(s) of theinvention as regulators of energy metabolism in mammals. For example,transcripts of IHKPK2 and IHKPK1 are more restricted in neuronal tissuesand testis of mammals (FIGS. 6A and 7A, respectively). IHKPK3transcripts show high expression in muscle and heart tissues (FIG. 5A).In addition, IHKPK1 and IHKPK3 are also clearly expressed in whiteadipose tissue (WAT) and brown adipose tissue (BAT); IHKPK2 shows highlevels of expression especially in WAT (see FIG. 5A, FIG. 6A, FIG. 7A,respectively). Brown adipose tissue is a well characterized tissue whichis well developed in newborn mammals, including humans. One importanttask of BAT is to generate heat and maintain body temperaturehomeostasis in newborn. Thus an expression of the protein of theinvention in adipose tissues is confirming a role in the regulation ofenergy homeostasis and thermogenesis.

Further, we show that the proteins of the invention are regulated byfasting and by genetically induced obesity. In this invention, we usedmouse models of insulin resistance and/or diabetes, such as micecarrying gene knockouts in the leptin pathway (for example, ob (leptin)or db (leptin receptor) mice) to study the expression of the protein ofthe invention. Such mice develop typical symptoms of diabetes, showhepatic lipid accumulation and frequently have increased plasma lipidlevels (see Bruning et al, 1998, Mol. Cell. 2:449-569). We found, forexample, that the expression of IHKPK1 and IHKPK2 is stronglyupregulated in the pancreas of fasted mice (see FIG. 6B and FIG. 7B,respectively). The expression of IHKPK3 is strongly upregulated in liverof fasted mice (FIG. 5B). In addition, a marked upregulation of IHKPK3,IHKPK1, and IHKPK2 can be observed in the metabolically active tissue(for example, brown adipose tissue (BAT)) of genetically obese (ob/ob)as well as of fasted mice (see FIG. 5B, FIG. 6B, and FIG. 7B,respectively). IHKPK3 is downregulated in white adipose tissue (WAT) offasted mice (see FIG. 5B), supporting a hypothesis that the protein ofthe invention is a modulator of adipogenesis.

In addition, we show in this invention that the mRNA of IHKPK1 andIHKPK2 and also RYK kinase is significantly down-regulated duringadipocyte differentiation in vitro (see EXAMPLES for more detail, seeFIG. 6C and FIG. 6C and FIG. 11), suggesting a role as modulator ofadipocyte lipid accumulation. With regard to changes in expressionintensity during the differentiation of preadipocytes to adipocytes, astrong increase in relative signal intensity can be observed for IHKPK3expression during the in vitro differentiation program of 3T3-L1 cells(see FIG. 5C). Thus, we conclude that the protein of the invention (orvariants thereof) have a function in the metabolism of mature mammalianadipocytes.

The invention also encompasses polynucleotides that encode Inositolhexakisphosphate kinase or RYK kinase or homologous proteins.Accordingly, any nucleic acid sequence, which encodes the amino acidsequences of Inositol hexakisphosphate kinase or RYK kinase can be usedto generate recombinant molecules that express Inositol hexakisphosphatekinase or RYK kinase. In a particular embodiment, the inventionencompasses the polynucleotide of (i) Drosophila CG10082, or humanIHKPK3, human IHKPK1, or human IHKPK2, or (ii) Drosophila dnt CG17559,or human RYK kinase. It will be appreciated by those skilled in the artthat as a result of the degeneracy of the genetic code, a multitude ofnucleotide sequences encoding Inositol hexakisphosphate kinase or RYKkinase, some bearing minimal homology to the nucleotide sequences of anyknown and naturally occurring gene, may be produced. Thus, the inventioncontemplates each and every possible variation of nucleotide sequencethat could be made by selecting combinations based on possible codonchoices.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridising to the claimed nucleotide sequences, and inparticular, those of the polynucleotide encoding (i) Drosophila CG10082,and/or human IHKPK3, IHKPK1, or IHKPK2, or (ii) Drosophila dnt CG17559,and/or human RYK kinase, under various conditions of stringency.Hybridisation conditions are based on the melting temperature (Tm) ofthe nucleic acid-binding complex or probe, as taught in Wahl, G. M. andS. L. Berger (1987: Methods Enzymol. 152:399-407) and Kimmel, A. R.(1987; Methods Enzymol. 152:507-511), and may be used at a definedstringency. Preferably, hybridization under stringent conditions meansthat after washing for 1 h with 1×SSC and 0.1% SDS at 50° C., preferablyat 55° C., more preferably at 62° C. and most preferably at 68° C.,particularly for 1 h in 0.2×SSC and 0.1% SDS at 50° C., preferably at55° C., more preferably at 62° C. and most preferably at 68° C., apositive hybridization signal is observed. Altered nucleic acidsequences encoding Inositol hexakisphosphate kinase or RYK kinase whichare encompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent Inositolhexakisphosphate kinase or RYK kinase.

The encoded proteins may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent Inositol hexakisphosphate kinase orRYK kinase. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of Inositol hexakisphosphate kinase or RYKkinase is retained. Furthermore, the invention relates to peptidefragments of the proteins or derivatives thereof such as cyclicpeptides, retro-inverso peptides or peptide mimetics having a length ofat least 4, preferably at least 6 and up to 50 amino acids.

Also included within the scope of the present invention are alleles ofthe genes encoding Inositol hexakisphosphate kinase or RYK kinase. Asused herein, an “allele” or “allelic sequence” is an alternative form ofthe gene, which may result from at least one mutation in the nucleicacid sequence. Alleles may result in altered mRNAs or polypeptides whosestructures or function may or may not be altered. Any given gene mayhave none, one, or many allelic forms. Common mutational changes, whichgive rise to alleles, are generally ascribed to natural deletions,additions, or substitutions of nucleotides. Each of these types ofchanges may occur alone, or in combination with the others, one or moretimes in a given sequence.

The nucleic acid sequences encoding Inositol hexakisphosphate kinase orRYK kinase may be extended utilising a partial nucleotide sequence andemploying various methods known in the art to detect upstream sequencessuch as promoters and regulatory elements. For example, one method whichmay be employed, “restriction-site” PCR, uses universal primers toretrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993)PCR Methods Applic. 2:318-322). Inverse PCR may also be used to amplifyor extend sequences using divergent primers based on a known region(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent to a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (PCR Methods Applic. 1:111-119).Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and libraries to walk ingenomic DNA (Clontech, Palo Alto, Calif.). This process avoids the needto screen libraries and is useful in finding intron/exon junctions.

In order to express a biologically active Inositol hexakisphosphatekinase or RYK kinase, the nucleotide sequences encoding Inositolhexakisphosphate kinase or RYK kinase or functional equivalents, may beinserted into appropriate expression vectors, i.e. a vector, whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods, which are well known to thoseskilled in the art, may be used to construct expression vectorscontaining sequences encoding Inositol hexakisphosphate kinase or RYKkinase and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques.synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook, J. et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

A variety of expression vector/host systems may be utilised to containand express sequences encoding Inositol hexakisphosphate kinase or RYKkinase. These include, but are not limited to, micro-organisms such asbacteria transformed with recombinant bacteriophage, plasmid, or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.baculovirus); plant cell systems transformed with virus expressionvectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)or with bacterial expression vectors (e.g. Ti or PBR322 plasmids); oranimal cell systems. The “control elements” or “regulatory sequences”are those non-translated regions of the vector-enhancers, promoters, 5′and 3′ untranslated regions which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilised, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used.

The presence of polynucleotide sequences encoding Inositolhexakisphosphate kinase and/or RYK kinase can be detected by DNA-DNA orDNA-RNA hybridisation or amplification using probes or portions orfragments of polynucleotides encoding Inositol hexakisphosphate kinaseor RYK kinase. Nucleic acid amplification based assays involve the useof oligonucleotides or oligomers based on a Inositol hexakisphosphatekinase or RYK kinase nucleic acid sequence to detect transformantscontaining DNA or RNA encoding Inositol hexakisphosphate kinase or RYKkinase. As used herein “oligonucleotides” or “oligomers” refer to anucleic acid sequence of at least about 10 nucleotides and as many asabout 60 nucleotides, preferably about 15 to 30 nucleotides, and morepreferably about 20-25 nucleotides, which can be used as a probe oramplimer.

A variety of protocols for detecting and measuring the expression ofInositol hexakisphosphate kinase or RYK kinase, using either polyclonalor monoclonal antibodies specific for the protein are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radio-immunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilising monoclonal antibodiesreactive to two non-interfering epitopes on Inositol hexakisphosphatekinase or Drl kinase is preferred, but a competitive binding assay maybe employed. These and other assays are described, among other places,in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual,APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to Inositol hexakisphosphate kinase orRYK kinase polynucleotides include oligo-labelling, nick translation,end-labelling or PCR amplification using a labelled nucleotide.

Alternatively, a Inositol hexakisphosphate kinase or RYK kinasesequence, or any portion thereof may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesise RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labelled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding Inositolhexakisphosphate kinase or RYK kinase may be cultured under conditionssuitable for the expression and recovery of the protein from cellculture. The protein produced by a recombinant cell may be secreted orcontained intracellularly depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing polynucleotides which encode Inositolhexakisphosphate kinase or RYK kinase may be designed to contain signalsequences, which direct secretion of Inositol hexakisphosphate kinase orRYK kinase through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encodingInositol hexakisphosphate kinase or RYK kinase to nucleotide sequenceencoding a polypeptide domain, which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilisedmetals, protein A domains that allow purification on immobilisedimmunoglobulin, and the domain utilised in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.) The inclusion ofcleavable linker sequences such as those specific for Factor XA orEnterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and Inositol hexakisphosphate kinase or RYK kinase may be used tofacilitate purification.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids andproteins of the invention and effector molecules thereof are useful indiagnostic and therapeutic applications implicated, for example but notlimited to, in metabolic disorders such as obesity as well as relateddisorders such as metabolic syndrome, eating disorder, cachexia,diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, and gallstones.Hence, diagnostic and therapeutic uses for the Inositol hexakisphosphatekinase or RYK kinase nucleic acids and proteins of the invention are,for example but not limited to, the following: (i) protein therapeutic,(ii) small molecule drug target, (iii) antibody target (therapeutic,diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/orprognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi)research tools, and (vii) tissue regeneration in vitro and in vivo(regeneration for all these tissues and cell types composing thesetissues and cell types derived from these tissues).

The nucleic acids and proteins of the invention are useful in diagnosticand therapeutic applications implicated in various applications asdescribed below. For example, but not limited to, cDNAs encoding theInositol hexakisphosphate kinase or RYK kinase proteins of the inventionand particularly their human homologues may be useful in gene therapy,and the Inositol hexakisphosphate kinase or RYK kinase proteins of theinvention and particularly their human homologues may be useful whenadministered to a subject in need thereof. By way of non-limitingexample, the compositions of the present invention will have efficacyfor treatment of patients suffering from, for example, but not limitedto, in metabolic disorders as described above.

The nucleic acids or fragments thereof, may further be useful indiagnostic applications, wherein the presence or amount of the nucleicacids or the proteins are to be assessed. These materials are furtheruseful in the generation of antibodies that bind immunospecifically tothe novel substances of the invention for use in therapeutic ordiagnostic methods.

For example, in one aspect, antibodies which are specific for Inositolhexakisphosphate kinase or RYK kinase may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which expressInositol hexakisphosphate kinase or RYK kinase. The antibodies may begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments, and fragments produced by a Fab expressionlibrary. Neutralising antibodies (i.e. those which inhibit dimerformation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunised by injectionwith Inositol hexakisphosphate kinase or RYK kinase any fragment oroligopeptide thereof which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminium hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in human, BCG (Bacille Calmette-Guerin) and Corynebacterium parvumare especially preferable. It is preferred that the peptides, fragments,or oligopeptides used to induce antibodies to Inositol hexakisphosphatekinase or RYK kinase have an amino acid sequence consisting of at leastfive amino acids, and more preferably at least 10 amino acids.

Monoclonal antibodies to Inositol hexakisphosphate kinase or RYK kinasemay be prepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Köhler, G. et al.(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. Proc. Natl. Acad. Sci. 80:2026-2030; Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceInositol hexakisphosphate kinase or RYK kinase-specific single chainantibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3). Antibodies may also be produced by inducing invivo production in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl.Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Fragments of antibodies against Inositol hexakisphosphate kinase or RYKkinase, which contain specific binding sites for Inositolhexakisphosphate kinase or RYK kinase, may also be generated. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragments which can be produced by Pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of F(ab′)₂ fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse, W. D. etal. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between Inositol hexakisphosphate kinase or RYK kinase and itsspecific antibody. A two-site, monoclonal-based immunoassay utilisingmonoclonal antibodies reactive to two non-interfering Inositolhexakisphosphate kinase or RYK kinase epitopes is preferred, but acompetitive binding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingInositol hexakisphosphate kinase or RYK kinase, or any fragment thereof,or nucleic acid effector molecules such as antisense molecules,aptamers, RNAi molecules or ribozymes may be used for therapeuticpurposes. In one aspect, aptamers, i.e. nucleic acid molecules, whichare capable of binding to a protein of the invention and modulating itsactivity, may be generated by a screening and selection procedureinvolving the use of combinatorial nucleic acid libraries.

In a further aspect, antisense molecules to the polynucleotide encodingInositol hexakisphosphate kinase or RYK kinase may be used in situationsin which it would be desirable to block the transcription of the mRNA.In particular, cells may be transformed with sequences complementary topolynucleotides encoding Inositol hexakisphosphate kinase or RYK kinase.Thus, antisense molecules may be used to modulate Inositolhexakis-phosphate kinase or RYK kinase activity, or to achieveregulation of gene function. Such technology is now well know in theart, and sense or antisense oligomers or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding Inositol hexakisphosphate kinase or RYK kinase.Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,can be used to construct recombinant vectors, which will expressantisense molecules complementary to the polynucleotides of a geneencoding Inositol hexakisphosphate kinase or RYK kinase. Thesetechniques are described both in Sambrook et al. (supra) and in Ausubelet al. (supra). Genes encoding Inositol hexakisphosphate kinase or RYKkinase can be turned off by transforming a cell or tissue withexpression vectors which express high levels of polynucleotide orfragment thereof which encodes Inositol hexakisphosphate kinase or RYKkinase. Such constructs may be used to introduce untranslatable sense orantisense sequences into a cell. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules untilthey are disabled by endogenous nucleases. Transient expression may lastfor a month or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or nucleic acid analogues suchas PNA, to the control regions of a gene encoding Inositolhexakisphosphate kinase or RYK kinase, i.e. the promoters, enhancers,and introns. Oligonucleotides derived from the transcription initiationsite, e.g. between positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using “triple helix”base-pairing methodology. Triple helix pairing is useful because itcause inhibition of the ability of the double helix to open sufficientlyfor the binding of polymerases, transcription factors, or regulatorymolecules. Recent therapeutic advances using triplex DNA have beendescribed in the literature (Gee, J. E. et al. (1994) In; Huber, B. E.and B. I. Carr, Molecular and Immunologic Approaches, Futura PublishingCo., Mt. Kisco, N.Y.). The antisense molecules may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyse thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyseendonucleolytic cleavage of sequences encoding Inositol hexakisphosphatekinase or RYK kinase. Specific ribozyme cleavage sites within anypotential RNA target are initially identified by scanning the targetmolecule for ribozyme cleavage sites which include the followingsequences: GUA, GUU, and GUC. Once identified, short RNA sequences ofbetween 15 and 20 ribonucleotides corresponding to the region of thetarget gene containing the cleavage site may be evaluated for secondarystructural features which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridisation with complementary oligonucleotides usingribonuclease protection assays.

Nucleic acid effector molecules, e.g. antisense molecules and ribozymesof the invention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesising oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding Inositol hexakisphosphate kinase or RYK kinase. Such DNAsequences may be incorporated into a variety of vectors with suitableRNA polymerase promoters such as T7 or SP6. Alternatively, these cDNAconstructs that synthesise antisense RNA constitutively or inducibly canbe introduced into cell lines, cells, or tissues. RNA molecules may bemodified to increase intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences at the 5′ and/or 3′ ends of the molecule or the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkageswithin the backbone of the molecule. This concept is inherent in theproduction of PNAs and can be extended in all of these molecules by theinclusion of non-traditional bases such as inosine, queosine, andwybutosine, as well as acetyl-, methyl-, thio-, and similarly modifiedforms of adenine, cytidine, guanine, thymine, and uridine which are notas easily recognised by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of Inositolhexakisphosphate kinase or RYK kinase, antibodies to Inositolhexakisphosphate kinase or RYK kinase, mimetics, agonists, antagonists,or inhibitors of Inositol hexakisphosphate kinase or RYK kinase. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilising compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones. The pharmaceutical compositionsutilised in this invention may be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which, can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g. by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilising processes. Afterpharmaceutical compositions have been prepared, they can be-placed in anappropriate container and labelled for treatment of an indicatedcondition. For administration of Inositol hexakisphosphate kinase or RYKkinase, such labelling would include amount, frequency, and method ofadministration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective does can beestimated initially either in cell culture assays, e.g. of preadipocytecell lines, or in animal models, usually mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. A therapeutically effective dose refers to that amount of activeingredient, for example Inositol hexakisphosphate kinase or RYK kinaseor fragments thereof, or antibodies against Inositol hexakisphosphatekinase or RYK kinase, which is effective against a specific condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or experimental animals, e.g.ED50 (the dose therapeutically effective in 50% of the population) andLD50 (the dose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions, whichexhibit large therapeutic indices, are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage being employed, the sensitivity of thepatient, and the route of administration. The exact dosage will bedetermined by the practitioner, in light of factors related to thesubject that requires treatment. Dosage and administration are adjustedto provide sufficient levels of the active moiety or to maintain thedesired effect. Factors, which may be taken into account, include theseverity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long-acting pharmaceutical compositionsmay be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation. Normal dosage amounts may vary from 0.1 to 100,000micrograms, up to a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

In another embodiment, antibodies which specifically bind Inositolhexakisphosphate kinase or RYK kinase may be used for the diagnosis ofconditions or diseases characterised by or associated with over- orunderexpression of Inositol hexakisphosphate kinase or RYK kinase, or inassays to monitor patients being treated with Inositol hexakisphosphatekinase or RYK kinase, agonists, antagonists or inhibitors. Theantibodies useful for diagnostic purposes may be prepared in the samemanner as those described above for therapeutics. Diagnostic assays forInositol hexakisphosphate kinase or RYK kinase include methods, whichutilise the antibody and a label to detect Inositol hexakisphosphatekinase or RYK kinase in human body fluids or extracts of cells ortissues. The antibodies may be used with or without modification, andmay be labelled by joining them, either covalently or non-covalently,with a reporter molecule. A wide variety of reporter molecules which areknown in the art may be used several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringInositol hexakisphosphate kinase or RYK kinase are known in the art andprovide a basis for diagnosing altered or abnormal levels of Inositolhexakisphosphate kinase or RYK kinase expression. Normal or standardvalues for Inositol hexakisphosphate kinase or RYK kinase expression areestablished by combining body fluids or cell extracts taken from normalmammalian subjects, preferably human, with antibody to Inositolhexakisphosphate kinase or RYK kinase under conditions suitable forcomplex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometric means.Quantities of Inositol hexakisphosphate kinase or RYK kinase expressedin control and disease, samples, e.g. from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease. Analysis of kinaseexpression could also be performed by determination of Inositolhexakisphosphate kinase or RYK kinase activity in assay formats wellknown in the art and described in more detail below.

In another embodiment of the invention, the Inositol hexakisphosphatekinase or RYK kinase polynucleotides may be used for diagnosticpurposes. The polynucleotides, which may be used, includeoligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.The polynucleotides may be used to detect and quantitate gene expressionin biopsied tissues in which expression of Inositol hexakisphosphatekinase or RYK kinase may be correlated with disease. The diagnosticassay may be used to distinguish between absence, presence, and excessexpression of Inositol hexakisphosphate kinase or RYK kinase, and tomonitor regulation of Inositol hexakisphosphate kinase or RYK kinaselevels during therapeutic intervention.

In one aspect, hybridisation with probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding Inositolhexakisphosphate kinase or RYK kinase or alleles thereof or closelyrelated molecules, may be used to identify nucleic acid sequences whichencode Inositol hexakisphosphate kinase or RYK kinase. The specificityof the probe, whether it is made from a highly specific region, e.g.unique nucleotides in the 5′ regulatory region, or a less specificregion, e.g. especially in the 3′ coding region, and the stringency ofthe hybridisation or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding Inositol hexakisphosphate kinase or RYK kinase,alleles, or related sequences. Probes may also be used for the detectionof related sequences, and should preferably contain at least 50% of thenucleotides from any of the Inositol hexakisphosphate kinase or RYKkinase encoding sequences. The hybridisation probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofa polynucleotide comprising (i) Drosophila CG10082, or IHKPK3, IHKPK1,or IHKPK2, or (ii) Drosophila dnt CG17559, or human RYK kinase, or froma genomic sequence including promoter, enhancer elements, and introns ofthe naturally occurring Inositol hexakisphosphate kinase or RYK kinasegene. Means for producing specific hybridisation probes for DNAsencoding Inositol hexakisphosphate kinase or RYK kinase include thecloning of nucleic acid sequences encoding Inositol hexakisphosphatekinase or RYK kinase derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesise RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labellednucleotides. Hybridisation probes may be labelled by a variety ofreporter groups; for example, radionuclides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

Polynucleotide sequences specific for Inositol hexakisphosphate kinaseor RYK kinase may be used for the diagnosis of conditions or diseases,which are associated with expression of Inositol hexakisphosphate kinaseor RYK kinase. Examples of such conditions or diseases include, but arenot limited to, pancreatic diseases and disorders, including diabetes.Polynucleotide sequences specific for Inositol hexakisphosphate kinaseor RYK kinase may also be used to monitor the progress of patientsreceiving treatment for pancreatic diseases and disorders, includingdiabetes. The polynucleotide sequences encoding Inositolhexakisphosphate kinase or RYK kinase may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilisingfluids or tissues from patient biopsies to detect altered Inositolhexakisphosphate kinase or RYK kinase expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding Inositolhexakisphosphate kinase or RYK kinase may be useful in assays thatdetect activation or induction of various metabolic diseases such asobesity as well as related disorders such as metabolic syndrome, eatingdisorder, cachexia, diabetes mellitus, hypertension, coronary heartdisease, hypercholesterolemia, dyslipidemia, osteoarthritis, andgallstones. The nucleotide sequences encoding Inositol hexakisphosphatekinase or RYK kinase may be labelled by standard methods, and added to afluid or tissue sample from a patient under conditions suitable for theformation of hybridisation complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. The presence of altered levels of nucleotidesequences encoding Inositol hexakisphosphate kinase or RYK kinase in thesample indicates the presence of the associated disease. Such assays mayalso be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or inmonitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of Inositol hexakisphosphate kinase or RYK kinase, a normalor standard profile for expression is established. This may beaccomplished by combining body fluids or cell extracts taken from normalsubjects, either animal or human, with a sequence, or a fragmentthereof, which encodes Inositol hexakisphosphate kinase or RYK kinase,under conditions suitable for hybridisation or amplification. Standardhybridisation may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease. Once disease is established and a treatment protocol isinitiated, hybridisation assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that, which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to metabolic diseases such as described above, the presenceof a relatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the metabolic, e.g. pancreatic diseases anddisorders.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding Inositol hexakisphosphate kinase or RYK kinase mayinvolve the use of PCR. Such oligomers may be chemically synthesised,generated enzymatically, or produced from a recombinant source.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′.fwdarw.3′) and another with antisense(3′.rarw.5′), employed under optimised conditions for identification ofa specific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantification of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of Inositolhexakisphosphate kinase or RYK kinase include radiolabelling orbiotinylating nucleotides, coamplification of a control nucleic acid,and standard curves onto which the experimental results are interpolated(Melby, P. C. et al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C.et al. (1993) Anal. Biochem. 212:229-236). The speed of quantificationof multiple samples may be accelerated by running the assay in an ELISAformat where the oligomer of interest is presented in various dilutionsand a spectrophotometric or colorimetric response gives rapidquantification.

In another embodiment of the invention, the nucleic acid sequences,specific for Inositol hexakisphosphate kinase or RYK kinase, may also beused to generate hybridisation probes, which are useful for mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome or to a specific region of the chromosome usingwell known techniques. Such techniques include FISH, FACS, or artificialchromosome constructions, such as yeast artificial chromosomes,bacterial artificial chromosomes, bacterial P1 constructions or singlechromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev.7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154. FISH (asdescribed in Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York, N.Y.) may be correlated with otherphysical chromosome mapping techniques and genetic map data. Examples ofgenetic map data can be found in the 1994 Genome Issue of Science(265:1981f). Correlation between the location of the gene encodingInositol hexakisphosphate kinase or RYK kinase on a physical chromosomalmap and a specific disease, or predisposition to a specific disease, mayhelp to delimit the region of DNA associated with that genetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier, or affectedindividuals. In situ hybridisation of chromosomal preparations andphysical mapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localised by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequences of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, the proteins, their catalytic orimmunogenic fragments or oligopeptides thereof, an in vitro model, agenetically altered cell or animal, can be used for screening librariesof compounds in any of a variety of drug screening techniques. One canidentify effectors, e.g. receptors, enzymes, proteins, ligands, orsubstrates that bind to, modulate or mimic the action of one or more ofthe proteins of the invention. The protein or fragment thereof employedin such screening may be free in solution, affixed to a solid support,borne on a cell surface, or located intracellularly. The formation ofbinding complexes, between the protein and the agent tested, may bemeasured. Agents could also, either directly or indirectly, influencethe activity of the proteins of the invention.

In vivo, the enzymatic kinase activity of the unmodified polypeptides ofInositol hexakisphosphate kinase or RYK kinase, or homologues thereoftowards a substrate can be measured. Activation of the kinases may beinduced in the natural context by extracellular or intracellularstimuli, such as signaling molecules or environmental influences. Onemay generate a system containing IHKPK or RYK kinases, or homologuesthereof, may it be an organism, a tissue, a culture of cells orcell-free environment, by exogenously applying this stimulus or bymimicking this stimulus by a variety of the techniques, some of themdescribed further below. A system containing activated Inositolhexakisphosphate kinase or RYK kinase, or homologues thereof may beproduced (i) for the purpose of diagnosis, study, prevention, andtreatment of diseases and disorders related to body-weight regulationand thermogenesis, for example, but not limited to, metabolic diseases,(ii) for the purpose of identifying or validating therapeutic candidateagents, pharmaceuticals or drugs that influence the genes of theinvention or their encoded polypeptides, (iii) for the purpose ofgenerating cell lysates containing activated polypeptides encoded by thegenes of the invention, (iv) for the purpose of isolating from thissource activated polypeptides encoded by the genes of the invention.

In one embodiment of the invention, one may produce activated Inositolhexakisphosphate kinase or RYK kinase independent of the natural stimulifor the above said purposes by, for example, but not limited to, (i) anagent that mimics the natural stimulus; (ii) an agent, that actsdownstream of the natural stimulus, such as activators of the Inositolhexakisphosphate kinase pathway or the RYK kinase, phorbol ester,anisomycin, constitutive active alleles of the Inositol hexakisphosphatekinase or RYK kinase itself as they are described or may be developed;(iii) by introduction of single or multiple amino acid substitutions,deletions or insertions within the sequence of Inositol hexakisphosphatekinase or RYK kinase to yield constitutive active forms; (iv) by the useof isolated fragments of Inositol hexakisphosphate kinase or RYK kinase.In addition, one may generate enzymatically active Inositolhexakisphosphate kinase or RYK kinase in an ectopic system, prokaryoticor eukaryotic, in vivo or in vitro, by co-transfering to this system theactivating components.

In addition activity of Inositol hexakisphosphate kinase or RYK kinaseagainst its physiological substrate(s) or derivatives thereof could bemeasured in cell-based assays. Agents may also interfere withposttranslational modifications of the protein, such as phosphorylationand dephosphorylation, farnesylation, palmitoylation, acetylation,alkylation, ubiquitination, proteolytic processing, subcellularlocalization and degradation. Moreover, agents could influence thedimerization or oligomerization of the proteins of the invention or, ina heterologous manner, of the proteins of the invention with otherproteins, for example, but not exclusively, docking proteins, enzymes,receptors, or translation factors. Agents could also act on the physicalinteraction of the proteins of this invention with other proteins, whichare required for protein function, for example, but not exclusively,their downstream signaling.

Methods for determining protein-protein interaction are well known inthe art. For example binding of a fluorescently labeled peptide derivedfrom the interacting protein to the protein of the invention, or viceversa, could be detected by a change in polarisation. In case that bothbinding partners, which can be either the full length proteins as wellas one binding partner as the full length protein and the other justrepresented as a peptide are fluorescently labeled, binding could bedetected by fluorescence energy transfer (FRET) from one fluorophore tothe other. In addition, a variety of commercially available assayprinciples suitable for detection of protein-protein interaction arewell known in the art, for example but not exclusively AlphaScreen(PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham.Alternatively, the interaction of the proteins of the invention withcellular proteins could be the basis for a cell-based screening assay,in which both proteins are fluorescently labeled and interaction of bothproteins is detected by analysing cotranslocation of both proteins witha cellular imaging reader, as has been developed for example, but notexclusively, by Cellomics or EvotecOAI. In all cases the two or morebinding partners can be different proteins with one being the protein ofthe invention, or in case of dimerization and/or oligomerization theprotein of the invention itself. Proteins of the invention, for whichone target mechanism of interest, but not the only one, would be suchprotein/protein interaction are Inositol hexakisphosphate kinase or RYKkinase.

Assays for determining enzymatic activity of the proteins of theinvention are well known in the art.

Of particular interest are screening assays for agents that have a lowtoxicity for mammalian cells. The term “agent” as used herein describesany molecule, e.g. protein or pharmaceutical, with the capability ofaltering or mimicking the physiological function of one or more of theproteins of the invention. Candidate agents encompass numerous chemicalclasses, though typically they are organic molecules, preferably smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 Daltons. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise carbocyclic orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups.

Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof. Candidateagents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Where the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal.

Candidate agents may also be found in kinase assays where a kinasesubstrate such as a protein, a peptide, a lipid, or an organic compound,which may or may not include modifications as further described below,or others are phosphorylated by the proteins or protein fragments of theinvention. A therapeutic candidate agent may be identified by itsability to increase or decrease the enzymatic activity of the proteinsof the invention. The kinase activity may be detected by change of thechemical, physical or immunological properties of the substrate due tophosphorylation. One example could be the transfer of radioisotopicallylabelled phosphate groups from an appropriate donor molecule to thekinase substrate catalyzed by the polypeptides of the invention. Thephosphorylation of the substrate may be followed by detection of thesubstrates autoradiography with techniques well known in the art.

In addition the generation of ATP by Inositol hexakisphosphate kinasecan be measured by luciferase-dependent bioluminescence generation, anassay principle well known in the art (ATP Determination Kit based onthis principle are offered by commercial suppliers).

Yet in another example, the change of mass of the substrate due to itsphosphorylation may be detected by mass spectrometry techniques. Onecould also detect the phosphorylation status of a substrate with ananalyte discriminating between the phosphorylated and unphosphorylatedstatus of the substrate. Such an analyte may act by having differentaffinities for the phosphorylated and unphosphorylated forms of thesubstrate or by having specific affinity for phosphate groups. Such ananalyte could be, but is not limited to, an antibody or antibodyderivative, a recombinant antibody-like structure, a protein, a nucleicacid, a molecule containing a complexed metal ion, an anion exchangechromatography matrix, an affinity chromatography matrix or any othermolecule with phosphorylation dependend selectivity towards thesubstrate.

Such an analyte could be employed to detect the kinase substrate, whichis immobilized on a solid support during or after an enzymatic reaction.If the analyte is an antibody, its binding to the substrate could bedetected by a variety of techniques as they are described in Harlow andLane, 1998, Antibodies, CSH Lab Press, NY. If the analyte molecule isnot an antibody, it may be detected by virtue of its chemical, physicalor immunological properties, being endogenously associated with it orengineered to it.

Yet in another example the kinase substrate may have features, designedor endogenous, to facilitate its binding or detection in order togenerate a signal that is suitable for the analysis of the substratesphosphorylation status. These features may be, but are not limited to, abiotin molecule or derivative thereof, a glutathione-S-transferasemoiety, a moiety of six or more consecutive histidine residues, an aminoacid sequence or hapten to function as an epitope tag, a fluorochrome,an enzyme or enzyme fragment. The kinase substrate may be linked tothese or other features with a molecular spacer arm to avoid sterichindrance.

In one example, the kinase substrate may be labelled with afluorochrome. The binding of the analyte to the labelled substrate insolution may be followed by the technique of fluorescence polarizationas it is described in the literature (see, for example, Deshpande, S. etal. (1999) Prog. Biomed. Optics (SPIE) 3603:261; Parker, G. J. et al.(2000) J. Biomol. Screen. 5:77-88; Wu, P. et al. (1997) Anal. Biochem.249:29-36). In a variation of this example, a fluorescent tracermolecule may compete with the substrate for the analyte to detect kinaseactivity by a technique which is known to those skilled in the art asindirect fluorescence polarization.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to Inositol hexakisphosphatekinase or RYK kinase large numbers of different small test compounds,e.g. aptamers, peptides, low-moleular weight compounds etc. aresynthesised on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with Inositol hexakisphosphatekinase or RYK kinase, or fragments thereof, and washed. Bound Inositolhexakisphosphate kinase or RYK kinase are then detected by methods wellknown in the art. Purified Inositol hexakisphosphate kinase or RYKkinase can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralising antibodies can be used to capture the peptide andimmobilise it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralising antibodies capable of binding Inositolhexakisphosphate kinase or RYK kinase specifically compete with a testcompound for binding Inositol hexakisphosphate kinase or RYK kinase. Inthis manner, the antibodies can be used to detect the presence of anypeptide, which shares one or more antigenic determinants with Inositolhexakisphosphate kinase or RYK kinase.

In additional embodiments, the nucleotide sequences which encodeInositol hexakisphosphate kinase or RYK kinase may be used in anymolecular biology techniques that have yet to be developed, provided thenew techniques rely on properties of nucleotide that are currentlyknown, including, but not limited to, such properties as the tripletgenetic code and specific base pair interactions.

The nucleic acids encoding the proteins of the invention can be used togenerate transgenic cell lines and animals. These transgenic non-humananimals are useful in the study of the function and regulation of theproteins of the invention in vivo. Transgenic animals, particularlymammalian transgenic animals, can serve as a model system for theinvestigation of many developmental and cellular processes common tohumans. A variety of non-human models of metabolic disorders can be usedto test modulators of the protein of the invention. Misexpression (forexample, overexpression or lack of expression) of the protein of theinvention, particular feeding conditions, and/or administration ofbiologically active compounts can create models of metablic disorders.

In one embodiment of the invention, such assays use mouse models ofinsulin resistance and/or diabetes, such as mice carrying gene knockoutsin the leptin pathway (for example, ob (leptin) or db (leptin receptor)mice). Such mice develop typical symptoms of diabetes, show hepaticlipid accumulation and frequently have increased plasma lipid levels(see Bruning et al, 1998, Mol. Cell. 2:449-569). Susceptible wild typemice (for example C57BI/6) show similiar symptoms if fed a high fatdiet. In addition to testing the expression of the proteins of theinvention in such mouse strains (see EXAMPLES), these mice could be usedto test whether administration of a candidate modulator alters forexample lipid accumulation in the liver, in plasma, or adipose tissuesusing standard assays well known in the art, such as FPLC, colorimetricassays, blood glucose level tests, insulin tolerance tests and others.

Transgenic animals may be made through homologous recombination inembryonic stem cells, where the normal locus of the gene encoding theprotein of the invention is mutated. Alternatively, a nucleic acidconstruct encoding the protein is injected into oocytes and is randomlyintegrated into the genome. One may also express the genes of theinvention or variants thereof in tissues where they are not normallyexpressed or at abnormal times of development. Furthermore, variants ofthe genes of the invention like specific constructs expressinganti-sense molecules or expression of dominant negative mutations, whichwill block or alter the expression of the proteins of the invention maybe randomly integrated into the genome. A detectable marker, such as lacZ or luciferase may be introduced into the locus of the genes of theinvention, where upregulation of expression of the genes of theinvention will result in an easily detectable change in phenotype.Vectors for stable integration include plasmids, retroviruses and otheranimal viruses, yeast artificial chromosomes (YACs), and the like. DNAconstructs for homologous recombination will contain at least portionsof the genes of the invention with the desired genetic modification, andwill include regions of homology to the target locus. Conveniently,markers for positive and negative selection are included. DNA constructsfor random integration do not need to contain regions of homology tomediate recombination. DNA constructs for random integration willconsist of the nucleic acids encoding the proteins of the invention, aregulatory element (promoter), an intron and a poly-adenylation signal.Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the field. For embryonic stem (ES)cells, an ES cell line may be employed, or embryonic cells may beobtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Suchcells are grown on an appropriate fibroblast-feeder layer and are grownin the presence of leukemia inhibiting factor (LIF). ES or embryoniccells may be transfected and can then be used to produce transgenicanimals. After transfection, the ES cells are plated onto a feeder layerin an appropriate medium. Cells containing the construct may be selectedby employing a selection medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Colonies that are positive may then be used for embryomanipulation and morula aggregation. Briefly, morulae are obtained from4 to 6 week old superovulated females, the Zona Pellucida is removed andthe morulae are put into small depressions of a tissue culture dish. TheES cells are trypsinized, and the modified cells are placed into thedepression closely to the morulae. On the following day the aggregatesare transfered into the uterine horns of pseudopregnant females. Femalesare then allowed to go to term. Chimeric offsprings can be readilydetected by a change in coat color and are subsequently screened for thetransmission of the mutation into the next generation (F1-generation).Offspring of the F1-generation are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogenic or congenic grafts or transplants, or in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimal, domestic animals, etc., for example, mouse, rat, guinea pig,sheep, cow, pig, and others. The transgenic animals may be used infunctional studies, drug screening, and other applications and areuseful in the study of the function and regulation of the proteins ofthe invention in vivo.

Finally, the invention relates to a kit comprising at least one of

-   -   (a) an Inositol hexakisphosphate kinase or RYK kinase nucleic        acid molecule or a fragment thereof;    -   (b) a vector comprising the nucleic acid of (a);    -   (c) a host cell comprising the nucleic acid of (a) or the vector        of (b);    -   (d) a polypeptide encoded by the nucleic acid of (a);    -   (e) a fusion polypeptide encoded by the nucleic acid of (a);    -   (f) an antibody, an aptamer or another receptor against the        nucleic acid of (a) or the polypeptide of (d) or (e) and    -   (g) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or forscreening applications as described above. The kit may furthercontainuser instructions.

The Figures show:

FIG. 1 shows the increase of triglyceride content of EP(2)0712 fliescaused by homozygous viable integration of the P-vector 5′ into the cDNAof Drosophila gene with GadFly Accession Number CG10082 (column 2; incomparison to controls without integration of this vector, column 1).

FIG. 2 shows the molecular organisation of the mutated inositolhexakisphosphate kinase (Gadfly Accession Number CG10082) gene locus.

FIG. 3 shows the IHKPK sequences

FIG. 3A shows the BLASTP search result for Gadfly Accession NumberCG10082 (Query) with the best human homolog matches (Sbject)

FIG. 3B. shows the nucleic acid sequence of human IHKPK3 (SEQ ID NO:1;GenBank Accession Number AF393812)

FIG. 3C. shows the amino acid sequence of human IHKPK3 (SEQ ID NO:2;GenBank Accession Number AAL17053)

FIG. 3D. shows the nucleic acid sequence of human IHKPK1 (SEQ ID NO:3;GenBank Accession Number NM_(—)153273 (formerly GenBank Accession No.BC012944)

FIG. 3E. shows the amino acid sequence of human IHKPK1 (SEQ ID NO:4;GenBank Accession Number NP_(—)695005 (formerly GenBank Accession No.AAH12944)

FIG. 3F. shows the nucleic acid sequence of human IHKPK2 (SEQ ID NO:5;GenBank Accession Number NM_(—)016291)

FIG. 3G. shows the amino acid sequence of human IHKPK2 (SEQ ID NO:6;GenBank Accession Number NP_(—)057375)

FIG. 3H shows a CLUSTAL W (1.82) multiple amino acid sequence alignmentof the Drosophila CG10082 (referred to as CG10082_Dm) sequence and thehuman inositol hexakisphosphate kinase 3 (referred to as IHPK3_Hs),human inositol hexakisphosphate kinase 1 (referred to as IHPK1_Hs), andhuman inositol hexakisphosphate kinase 2 (referred to as IHPK2_Hs). Thealignment was produced using the multisequence alignment program ofClustal W software (Higgins D. G. and Sharp P. M., (1989) CABIOS 5(2):151-153. Identical amino acid residues are marked with a star.

FIG. 4 shows the sequences of the mouse inositol hexakisphosphate kinase3 (IHKPK3)

FIG. 4A shows the cDNA of the mouse IHKPK3 (SEQ ID NO: 7).

FIG. 4B shows the amino acid sequence (one letter code) encoding themouse IHKPK3 protein (SEQ ID NO: 8).

FIG. 5-7. Expression of IHKPK in mammalian tissues. The relativeRNA-expression is shown on the X-axis. In FIGS. A and B, the tissuestested are given on the Y-axis. “WAT” refers to white adipose tissue,“BAT” refers to brown adipose tissue. In FIG. C, the Y-axis representsthe time axis. “d0” refers to day 0 (start of the experiment),“d2”-“d10” refers to day 2-day 10 of adipocyte differentiation).

FIG. 5. Expression of IHKPK3 in mammalian tissues.

FIG. 5A: Real-time PCR analysis of IHKPK3 in wildtype mouse tissues.

FIG. 5B: Real-time PCR mediated analysis of IHKPK3 in different mousemodels.

FIG. 5C: Real-time PCR mediated comparison of IHKPK3 expression duringthe differentiation of 3T3-L1 cells from preadipocytes to matureadipocytes.

FIG. 6. Expression of IHKPK1 in mammalian tissues.

FIG. 6A: Real-time PCR analysis of IHKPK1 in wildtype mouse tissues.

FIG. 6B: Real-time PCR mediated analysis of IHKPK1 in different mousemodels.

FIG. 6C: Real-time PCR mediated comparison of IHKPK1 expression duringthe differentiation of 3T3-L1 cells from preadipocytes to matureadipocytes.

FIG. 7. Expression of IHKPK2 in mammalian tissues.

FIG. 7A: Real-time PCR analysis of IHKPK2 in wildtype mouse tissues.

FIG. 7B: Real-time PCR mediated analysis of IHKPK2 in different mousemodels.

FIG. 7C: Real-time PCR mediated comparison of IHKPK2 expression duringthe differentiation of 3T3-L1 cells from preadipocytes to matureadipocytes.

FIG. 8A shows the increase of triglyceride content of HD-EP(2)21861flies caused by homozygous viable integration of the P-vector into thefirst large intron of doughnut on 2 (in comparison to controls withoutintegration of this vector).

FIG. 8B shows the increase of triglyceride and glycogen contents of adrl (Gadfly Accession Number CG17348) mutant.

FIG. 9 shows the molecular organisation of the mutated dnt (GadflyAccession Number CG17559) gene locus.

FIG. 10A shows the BLASTP search result for CG17559 (Query) with thebest human homolog match (Sbject).

FIG. 10B. shows the nucleic acid sequence of RYK kinase (human RYKreceptor-like tyrosine kinase precursor, SEQ ID NO:9; GenBank AccessionNumber NM_(—)002958)

FIG. 10C. shows the amino acid sequence of RYK kinase (human RYKreceptor-like tyrosine kinase precursor, SEQ ID NO:10; GenBank AccessionNumber NP_(—)002949)

FIG. 11. Expression of RYK kinase in mammalian tissues. Real-time PCRmediated comparison of RYK kinase expression during the differentiationof 3T3-L1 cells from preadipocytes to mature adipocytes. The relativeRNA-expression is shown on the X-axis, the days of differentiation areshown on the Y-axis (d0=day 0, start of the experiment, until d10=day10).

The examples illustrate the invention:

EXAMPLE 1 Measurement of Energy Storage Metabolites Content

Mutant flies are obtained from a fly mutation stock collection. Theflies are grown under standard conditions known to those skilled in theart. In the course of the experiment, additional feedings with bakersyeast (Saccharomyces cerevisiae) are provided. The average increase ofenergy storage metabolites (triglyceride and glycogen) content ofDrosophila containing the EP-vectors in homozygous viable integrationwas investigated in comparison to control flies (see FIGS. 1 and 8). Fordetermination of triglyceride, flies were incubated for 5 min at 90° C.in an aqueous buffer using a waterbath, followed by hot extraction.After another 5 min incubation at 90° C. and mild centrifugation, thetriglyceride content of the flies extract was determined using SigmaTriglyceride (INT 336-10 or -20) assay by measuring changes in theoptical density according to the manufacturer's protocol. The glycogencontent of the flies extract was determined using Roche (StarchUV-method Cat. No. 0207748) assay by measuring changes in the opticaldensity according to the manufacturer's protocol. As a reference proteincontent of the same extract was measured using BIO-RAD DC Protein Assayaccording to the manufacturer's protocol. The assay was repeated severaltimes. The average triglyceride level of all flies of the EP collections(referred to as ‘EP-control’) is shown as 100% in FIGS. 1 and 8.

EP(2)0712 homozygous flies show constantly a higher triglyceride contentthan the controls (50%; column 2 in FIG. 1). Therefore, the loss of geneactivity in the locus 57F6 on chromosome 2R where the EP-vector ofHD-EP(2)0712 flies is homozygous viable integrated, is responsible forchanges in the metabolism of the energy storage triglycerides. Evenheterozygous integration of this vector into the cDNA of CG10082 mildlyelvevates the triglyceride content.

HD-EP(2)21861 homozygous flies show constantly a higher triglyceridecontent than the controls (70%; column 2 in FIG. 8A). Therefore, theloss of gene activity in the locus 37D4-37D6 on chromosome 2L where theEP-vector of HD-EP(2)21861 flies is homozygous viable integrated, isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing an model for obese flies.

HD-EP21688 homozygous flies show constantly a higher triglyceridecontent (than the controls (column 3 in FIG. 8B). HD-EP21688 homozygousflies also show a higher glycogen content than the controls (column 5 inFIG. 8B). The average glycogen level of an internal assay controlconsisting of two different wildtype strains and an inconspicuousEP-line of the HD stock collection is shown as 100% in the fourth columnin FIG. 8B. Standard deviations of the measurements are shown as thinbars. Therefore, the loss of gene activity is responsible for changes inthe metabolism of the energy storage metabolites.

EXAMPLE 2 Identification of Drosophila Genes and Proteins Associatedwith Metabolic Control

In FIG. 2, genomic DNA sequence is represented by the assembly as adotted black line (from position 16568000 to 16581500 on chromosome 2R)including the integration sites of vector for line EP(2)0712 and in FIG.9, genomic DNA sequence is represented by the assembly as a dotted blackline (from position 19160000 to 19195000 on chromosome 2L) including theintegration sites of vector for line HD-EP(2)21861. Transcribed DNAsequences (ESTs) and predicted exons are shown as bars in the lower twolines. Predicted exons of the cDNA with GadFly Accession Number CG10082(FIG. 2) or GadFly Accession Number CG17559 (FIG. 9) are shown as darkgrey bars and introns as light grey bars.

Inositol hexakisphosphate kinase encodes for a gene that is predicted byGadFly sequence analysis programs as Accession Number CG10082. dntencodes for a gene that is predicted by GadFly sequence analysisprograms as Accession Number CG17559. Public DNA sequence databases (forexample, NCBI GenBank) were screened thereby identifying thecorresponding integration sites that are causing an increase oftriglyceride content. For example, EP(2)0712 is integrated 5′ inantisense orientation of the cDNA with Accession Number CG10082. Inanother example, HD-EP(2)21861 is integrated into the first large intronof doughnut on 2 (dnt) in antisense orientation of the cDNA withAccession Number CG17559. Therefore, expression of the cDNA encodingAccession Number CG10082 or CG17559 could be effected by homozygousintegration of vectors, leading to increase of the energy storagetriglycerides.

EXAMPLE 3 Identification of Human Inositol Hexakisphosphate Kinase orRYK Kinase Homologues

Inositol hexakisphosphate kinase or RYK kinase homologous proteins andnucleic acid molecules coding therefore are obtainable from insect orvertebrate species, e.g. mammals or birds. Particularly preferred arenucleic acids and polypeptides encoded thereby of Drosophila CG10082,human inositol hexakisphosphate kinase 3 (GenBank Accession NumberAF393812 for the cDNA, AAL17053 for the protein, SEQ ID NO 1 and 2;FIGS. 3B and 3C, respectively), human inositol hexakisphosphate kinase 1(GenBank Accession Number NM_(—)153273 for the cDNA, NP_(—)695005 forthe protein, SEQ ID NO:3 and 4, FIGS. 3D and 3E, respectively), inositolhexakisphosphate kinase 2 (GenBank Accession Number NM_(—)016291 for thecDNA, NP_(—)057375 for the protein, SEQ ID NO:5 and 6, FIGS. 3F and 3G,respectively), or of Drosophila dnt CG17559, human RYK kinase (GenBankAccession Number NM_(—)002958 for the cDNA, NP_(—)002949 for theprotein, SEQ ID NO 9 and 10; FIGS. 10B and C, respectively).

As shown in FIG. 3A, the gene product of Drosophila CG10082 is 45%homologous to human IHKPK2 (Accession Number XM_(—)030060.1), which isencoded by sequences located between nucleotide 21459 and 29259 on humanchromosome 3 (Accession Number NT_(—)005990), and 51% homologous tohuman KIAA0263 protein (GenBank Accession Number XP_(—)055065). As shownin FIG. 10A, Drosophila CG17559 gene product is 55% homologous to humantyrosine-protein kinase RYK precursor (GenBank Accession NumberNP_(—)002949) (herein referred to as RYK kinase).

EXAMPLE 4 Cloning of the Mouse IHKPK3

The Mus musculus IHKPK3 was cloned by polymerase chain reaction on themouse cDNA clone (EST-clone) image: 336155 (ResGen InvitrogenCorporation) using proofreading DNA-Polymerase (Pfu Turbo, Stratagene)according to standard procedures known to those skilled in the art.

The following primers were used: mIHKPK3.for(attB1-Primer) (SEQ IDNO:11): 5′ GGGG ACA AGT TTG TAC AAA AAA GCA GGC T CTCGAG TT GGG AGG ACTTGG TGC CAT G 3′ and mIHKPK3.rev(attB2-Primer) (SEQ ID NO:12): 5′ GGGGAC CAC TTT GTA CAA GAA AGC TGG GT CTCGAG G TTC CAA GAA GCT TCA TTC TCC T3′. The primers contain a Gateway extension (in bold, Invitrogen) forfurther cloning.

EXAMPLE 5 Expression of the Polypeptides in Mammalian Tissues

For analyzing the expression of the polypeptides disclosed in thisinvention in mammalian tissues, several mouse strains (preferrably micestrains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standardmodel systems in obesity and diabetes research) were purchased fromHarlan Winkelmann (33178 Borchen, Germany) and maintained under constanttemperature (preferrably 22° C.), 40 per cent humidity and a light/darkcycle of preferrably 14/10 hours. The mice were fed a standard chow (forexample, from ssniff Spezialitäten GmbH, order number ssniff M-ZV1126-000). For the fasting experiment (“fasted wild type mice”), wildtype mice were starved for 48 h without food, but only water supplied adlibitum (see, for example, Schnetzler et al. J Clin Invest July1993;92(1):272-80, Mizuno et al. Proc Natl Acad Sci U S A Apr. 16,1996;93(8):3434-8). Animals were sacrificed at an age of 6 to 8 weeks.The animal tissues were isolated according to standard procedures knownto those skilled in the art, snap frozen in liquid nitrogen and storedat −80° C. until needed.

For analyzing the role of the proteins disclosed in this invention inthe in vitro differentiation of different mammalian cell culture cellsfor the conversion of pre-adipocytes to adipocytes, mammalian fibroblast(3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113-116, 1974) wereobtained from the American Tissue Culture Collection (ATCC, Hanassas,Va., USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described in the prior art (e.g., Qiu.et al., J. Biol. Chem. 276:11988-95, 2001; Slieker et al., BBRC 251:225-9, 1998). At various time points of the differentiation procedure,beginning with day 0 (day of confluence) and day 2 (hormone addition;for example, dexamethasone and 3-isobutyl-1-methylxanthine), up to 10days of differentiation, suitable aliquots of cells were taken every twodays.

Expression Analysis of the Proteins of the Invention

RNA was isolated from mouse tissues or cell culture cells using TrizolReagent (for example, from Invitrogen, Karlsruhe, Germany) and furtherpurified with the RNeasy Kit (for example, from Qiagen, Germany) incombination with an DNase-treatment according to the instructions of themanufacturers and as known to those skilled in the art. Total RNA wasreverse transcribed (preferrably using Superscript II RNaseH-ReverseTranscriptase, from Invitrogen, Karlsruhe, Germany) and subjected toTaqman analysis preferrably using the Taqman 2×PCR Master Mix (fromApplied Biosystems, Weiterstadt, Germany; the Mix contains according tothe Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems,Weiterstadt, Germany).

For the analysis of the expression of the transcripts of the proteins ofthe invention, taqman analysis was performed using the followingprimer/probe pairs:

For the amplification of IHKPK3:

Mouse IHKPK 3 forward primer (Seq ID NO: 13) 5′-AGC TTC TCC CTC TTG CCTTCC-3′; Mouse IHKPK 3 reverse primer (Seq ID NO: 14) 5′-GTC CGA GCT GTGCCG C-3′; Mouse IHKPK 3 Taqman probe (Seq ID NO: 15) (5/6-FAM) TGG GAGGAC TTG GTG CCA TGG TG (5/6-TAMRA)

30 For the amplification of IHKPK1:

Mouse IHKPK 1 forward primer (Seq ID NO: 16) 5′-GCA CAC AGC ACA TTC AAGGG-3′; Mouse IHKPK 1 reverse primer (Seq ID NO: 17) 5′-AGC CTC TGT CTGGCC CAT C-3′; Mouse IHKPK 1 Taqman probe (Seq ID NO: 18) (5/6-FAM) TTCCGG GAT GAC CCC ACT GTT CA (5/6-TAMRA)

For the amplification of IHKPK2:

Mouse IHKPK 2 forward primer (Seq ID NO: 19) 5′-GGT GCA GGG CTT CAA GGAG-3′; Mouse IHKPK 2 reverse primer (Seq ID NO: 20) 5′-AGC TCA CGG CGCAGG TAC-3′; Mouse IHKPK 2 Taqman probe (Seq ID NO: 21) (5/6-FAM) CGC TTTTCC AGT TCT TTC ACA ATG GGC (5/6-TAMRA) For the amplification of RYKkinase:

Mouse RYK kinase forward primer (Seq ID NO: 22) 5′-TGG GAG CCT ACG TCTGAC TTC-3′; Mouse RYK kinase reverse primer (Seq ID NO: 23) 5′-CAT CCGTGA CAG ACA GGC AC-3′; Mouse RYK kinase Taqman probe (Seq ID NO: 24)(5/6-FAM) CTC CCA GCT CCG CCA CTC AGA AGA (5/6-TAMRA)

Expression profiling studies confirm the particular relevance of IHKPK3,IHKPK1, IHKPK2, and RYK kinase as regulator of energy metabolism inmammals. The results are shown in FIGS. 5A, 6A, and 7A. IHKPK1 andIHKPK2 proteins show higher expression compared to IHKPK3. IHKPK3 showsits highest expression levels in muscle and heart tissues. In addition,significant expression levels of IHPKK3 were found in metabolic activetissues like white adipocyte tissue (WAT) and brown adipocyte tissue(BAT), (FIG. 5A), confirming a role in the regulation of energyhomeostasis and thermogenesis.

Further, we show that IHKPK3, IHKPK1, and IHKPK2 are regulated byfasting and by genetically induced obesity, and that thus the expressionof IHKPK3, IHKPK1, and IHKPK2 is under metabolic control. In thisinvention, we used mouse models of insulin resistance and/or diabetes,such as mice carrying gene knockouts in the leptin pathway (for example,ob (leptin) or db (leptin receptor/ligand) mice) to study the expressionof the protein of the invention. Such mice develop typical symptoms ofdiabetes, show hepatic lipid accumulation and frequently have increasedplasma lipid levels (see Bruning et al, 1998, Mol. Cell. 2:449-569). Forexample, in genetically induced obese (ob/ob) mice, expression of IHKPK3is strongely induced in BAT and liver. In addition, expression in WAT isseverely reduced of wildtype levels in fasted mice. A very stronginduction of IHKPK 3 in liver is also noted in fasted mice (FIG. 5B).

The IHKPK proteins were also examined in the in vitro differentiationmodels for the conversion of pre-adipocytes to adipocytes, as describedabove. As shown in FIG. 5C, IHKPK3 shows a strong induction of itsexpression during differentiation, starting on day 8 of differentiationin 3T3-L1 cells (up to 6-fold increase of expression; as shown in FIG.5C). As shown in FIG. 6C, the expression of IHKPK 1 is stronglydownregulated during differentiation, starting on day 4 ofdifferentiation in 3T3-L1 cells. The expression of IHKPK 2 is alsosignificantly downregulated during differentiation, starting on day 8 ofdifferentiation in 3T3-L1 cells (as shown in FIG. 7C).

Taqman analysis revealed that RYK kinase is ubiquitously expressed (datanot shown). RYK kinase is expressed in several cell culture modelsystems of adipocyte differentiation. During the differentiation of3T3-L1 cells, the level of expression of RYK kinase is decreased duringthe progression of these preadipocytes to mature adipocytes (FIG. 11).

EXAMPLE 6 In Vitro Assays for the Determination of Triglyceride Storage,Synthesis and Transport

Obesity is known to be caused by different reasons such as non-insulindependent diabetes, increase in triglycerides, increase in carbohydratebound energy and low energy expenditure. For example, an increase inenergy expenditure (and thus, lowering the body weight) would includethe elevated utilization of both circulating and intracellular glucoseand triglycerides, free or stored as glycogen or lipids as fuel forenergy and/or heat production. The cellular level of triglycerides andglycogen is analyzed in cells overexpressing the protein of theinvention.

Preparation of Cell Lysates for Analysis of Metabolites

Starting at confluence (d0), cell media was changed every 48 hours.Cells and media were harvested 8 hours prior to media change as follows.Media was collected, and cells were washed twice in PBS prior to lysesin 600 μl HB-buffer (0.5% polyoxyethylene 10 tridecylethane, 1 mM EDTA,0.01M NaH₂PO₄, pH 7.4). After inactivation at 70° C. for 5 minutes, celllysates were prepared on Bio 101 systems lysing matrix B (0.1 mm silicabeads; Q-Biogene, Carlsbad, USA) by agitation for 2×45 seconds at aspeed of 4.5 (Fastprep FP120, Bio 101 Thermosavant, Holbrock, USA).Supernatants of lysed cells were collected after centrifugation at 3000rpm for 2 minutes, and stored in aliquots for later analysis at −80° C.

Changes in Cellular Triglyceride Levels During Adipogenesis

Cell lysates and media were simultaneously analysed in 96-well platesfor total protein and triglyceride content using the Bio-Rad DC Proteinassay reagent (Bio-Rad, Munich, Germany) according to the manufacturer'sinstructions and a modified enzymatic triglyceride kit (GPO-Trinder;Sigma) briefly final volumes of reagents were adjusted to the 96-wellformat as follows: 10 μl sample was incubated with 200 μl reagent A for5 minutes at 37° C. After determination of glycerol (initial absorbanceat 540 nm), 50 μl reagent B was added followed by another incubation for5 minutes at 37° C. (final absorbance at 540 nm). Glycerol andtriglyceride concentrations were calculated using a glycerol standardset (Sigma) for the standard curve included in each assay.

Changes in Cellular Glycogen Levels During Adipogenesis

Cell lysates and media were simultaneously analysed in triplicates in96-well plates for total protein and glycogen content using the Bio-RadDC Protein assay reagent (Bio-Rad, Munich, Germany) according to themanufacturer's instructions and an enzymatic starch kit from Hoffmann-LaRoche (Basel, Switzerland). 10-μl samples were incubated with 20-μlamyloglucosidase solution for 15 minutes at 60° C. to digest glycogen toglucose. The glucose is further metabolised with 100 μl distilled waterand 100 μl of enzyme cofactor buffer and 12 μl of enzyme buffer(hexokinase and glucose phosphate dehydrogenase). Background glucoselevels are determined by subtracting values from a duplicate platewithout the amyloglucosidase. Final absorbance is determined at 340 nm.HB-buffer as blank, and a standard curve of glycogen (Hoffmann-La Roche)were included in each assay. Glycogen content in samples were calculatedusing a standard curve.

Synthesis of Lipids During Adipogenesis

During the terminal stage of adipogenesis (day 12) cells were analysedfor their ability to metabolise lipids. A modified protocol to themethod of Jensen et al (2000) for lipid synthesis was established. Cellswere washed 3 times with PBS prior to serum starvation inKrebs-Ringer-Bicarbonate-Hepes buffer (KRBH; 134 nM NaCl, 3.5 mM KCl,1.2 mM KH₂ PO₄, 0.5 mM MgSO₄,1.5 mM CaCl₂, 5 mM NaHCO₃, 10 mM Hepes, pH7.4), supplemented with 0.1% FCS for 2.5 h at 37° C. Forinsulin-stimulated lipid synthesis, cells were incubated with 1 μMbovine insulin (Sigma; carrier: 0.005N HCl) for 45 min at 37° C. Basallipid synthesis was determined with carrier only. ¹⁴C(U)-D-glucose (NENLife Sciences) in a final activity of 1 μCi/Well/ml in the presence of 5mM glucose was added for 30 min at 37° C. For the calculation ofbackground radioactivity, 25 μM cytochalasin B (Sigma) was used. Allassays were performed in duplicate wells. To terminate the reaction,cells were washed 3 times with ice cold PBS, and lysed in 1 ml 0.1 NNaOH. Protein concentration of each well was assessed using the standardBiuret method (Protein assay reagent; Bio-Rad). Total lipids wereseparated from aqueous phase after overnight extraction in Insta-Fluorscintillation cocktail (Packard Bioscience) followed by scintillationcounting.

Transport and Metabolism of Free Fatty Acids During Adipogenesis

During the terminal stage of adipogenesis (d12) cells were analysed fortheir ability to transport long chain fatty acid across the plasmamembrane. A modified protocol to the method of Abumrad et al (1991)(Proc. Natl. Acad. Sci. USA, 1991: 88; 6008-12) for cellulartransportation of fatty acid was established. In summary, cells werewashed 3 times with PBS prior to serum starvation. This was followed byincubation in KRBH buffer supplemented with 0.1% FCS for 2.5 h at 37° C.Uptake of exogenous free fatty acids was initiated by the addition ofisotopic media containing non radioactive oleate and (³H)oleate (NENLife Sciences) complexed to serum albumin in a final activity of 1μCi/Well/ml in the presence of 5 mM glucose for 30 min at roomtemperature (RT). For the calculation of passive diffusion (PD) in theabsence of active transport (AT) across the plasma membrane 20 mM ofphloretin in glucose free media (Sigma) was added for 30 min at RT. Allassays were performed in duplicate wells. To terminate the activetransport 20 mM of phloretin in glucose free media was added to thecells. Cells were lysed in 1 ml 0.1N NaOH and the protein concentrationof each well were assessed using the standard Biuret method (Proteinassay reagent; Bio-Rad). Esterified fatty acids were separated from freefatty acids using overnight extraction in Insta-Fluor scintillationcocktail (Packard Bioscience) followed by scintillation counting.

EXAMPLE 7 Glucose Uptake Assay

For the determination of glucose uptake, cells were washed 3 times withPBS prior to serum starvation in KRBH buffer supplemented with 0.1% FCSand 0.5 mM Glucose for 2.5 h at 37° C. For insulin-stimulated glucoseuptake, cells were incubated with 1 microM bovine insulin (Sigma;carrier: 0.005N HCl) for 45 min at 37° C. Basal glucose uptake wasdetermined with carrier only. Non-metabolizable 2-deoxy-3H-D-glucose(NEN Life Science, Boston, USA) in a final activity of 0.4 μCi/Well/mlwas added for 30 min at 37° C. For the calculation of backgroundradioactivity, 25 μM cytochalasin B (Sigma) was used. All assays wereperformed in duplicate wells. To terminate the reaction, cells werewashed 3 times with ice cold PBS, and lysed in 1 ml 0.1N NaOH. Proteinconcentration of each well was assessed using the standard Biuret method(Protein assay reagent; Bio-Rad), and scintillation counting of celllysates in 10 volumes Ultima-gold cocktail (Packard Bioscience,Groningen, Netherlands) was performed.

EXAMPLE 8 Generation and Analysis of Transgenic Mice

Generation of the Transgenic Animals

Mouse cDNA was isolated from mouse brown adipose tissue (BAT) usingstandard protocols as known to those skilled in the art. The cDNA wasamplified by RT-PCR and point mutations were introduced into the cDNA.

The resulting mutated cDNA was cloned into a suitable transgenicexpression vector. The transgene was microinjected into the malepronucleus of fertilized mouse embryos (preferably strain C57/BL6/CBA F1(Harlan Winkelmann). Injected embryos were transferred intopseudo-pregnant foster mice. Transgenic founders were detected by PCRanalysis. Two independent transgenic mouse lines containing theconstruct were established and kept on a C57/BL6 background. Briefly,founder animals were backcrossed with C57/BL6 mice to generate F1 micefor analysis. Transgenic mice were continously bred onto the C57/BI6background. The expression of the proteins of the invention can beanalyzed by taqman analysis as described above, and further analysis ofthe mice can be done as known to those skilled in the art.

All publications and patents mentioned in the above specification areherein incorporated by reference.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A pharmaceutical composition comprising a nucleic acid molecule ofthe Inositol hexakisphosphate kinase or RYK kinase gene family or apolypeptide encoded thereby or a fragment or a variant of said nucleicacid molecule or said polypeptide, or an effector of said nucleic acidmolecule or said polypeptide, e.g. an antibody, an aptamer or anotherreceptor recognizing a nucleic acid molecule of the Inositolhexakisphosphate kinase or RYK kinase gene family or said polypeptideencoded thereby, preferably together with pharmaceutically acceptablecarriers, diluents and/or adjuvants.
 2. The composition of claim 1,wherein the nucleic acid molecule is a vertebrate or insect Inositolhexakisphosphate kinase or RYK kinase nucleic acid, particulary encodinghuman inositol hexakisphosphate kinase (SEQ ID NO:1, SEQ ID NO:3, or SEQID NO: 5), human RYK kinase (SEQ ID NO: 9), or a fragment thereof or avariant thereof.
 3. The composition of claim 1 or 2, wherein saidnucleic acid molecule (a) hybridizes at 50° C. in a solution containing1×SSC and 0.1% SDS to a nucleic acid molecule as defined in claim 2and/or a nucleic acid molecule which is complementary thereto; (b) it isdegenerate with respect to the nucleic acid molecule of (a); (c) encodesa polypeptide which is at least 85%, preferably at least 90%, morepreferably at least 95%, more preferably at least 98% and up to 99.6%identical to human inositol hexakisphosphate kinase 3 (SEQ ID NO:2),human inositol hexakisphosphate kinase 1 (SEQ ID NO:4), human inositolhexakisphosphate kinase 2 (SEQ ID NO:6), or human RYK kinase (SEQ IDNO:10), as defined in claim 2; (d) differs from the nucleic acidmolecule of (a) to (c) by mutation and wherein said mutation causes analteration, deletion, duplication or premature stop in the encodedpolypeptide.
 4. The composition of any one of claims 1-3, wherein thenucleic acid molecule is a DNA molecule, particularly a cDNA or agenomic DNA.
 5. The composition of any one of claims 1-4, wherein saidnucleic acid encodes a polypeptide contributing to regulating the energyhomeostasis and/or the metabolism of triglycerides.
 6. The compositionof any one of claims 1-5, wherein said nucleic acid molecule is arecombinant nucleic acid molecule.
 7. The composition of any one ofclaims 1-6, wherein the nucleic acid molecule is a vector, particularlyan expression vector.
 8. The composition of any one of claims 1-5,wherein the polypeptide is a recombinant polypeptide.
 9. The compositionof claim 8, wherein said recombinant polypeptide is a fusionpolypeptide.
 10. The composition of any one of claims 1-7, wherein saidnucleic acid molecule is selected from hybridization probes, primers andanti-sense oligonucleotides.
 11. The composition of any one of claims1-10 which is a diagnostic composition.
 12. The composition of any oneof claims 1-10 which is a therapeutic composition.
 13. The compositionof any one of claims 1-12 for the manufacture of an agent for detectingand/or verifying, for the treatment, alleviation and/or prevention of andisorders, including metabolic diseases such as obesity and otherbody-weight regulation disorders as well as related disorders such asmetabolic syndrome, eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hyper-cholesterolemia,dyslipidemia, osteoarthritis, gallstones and others, in cells, cellmasses, organs and/or subjects.
 14. Use of a nucleic acid molecule ofthe Inositol hexakisphosphate 9 kinase or RYK kinase gene family or apolypeptide encoded thereby or a fragment or a variant of said nucleicacid molecule or said polypeptide or an antibody, an aptamer or anotherreceptor recognizing a nucleic acid molecule of the Inositolhexakisphosphate kinase or RYK kinase gene family or a polypeptideencoded thereby for controlling the function of a gene and/or a geneproduct which is influenced and/or modified by an Inositolhexakisphosphate kinase or RYK kinase homologous polypeptide.
 15. Use ofa nucleic acid molecule of the Inositol hexakisphosphate kinase or RYKkinase gene family or a polypeptide encoded thereby or a fragment or avariant of said nucleic acid molecule or said polypeptide or anantibody, an aptamer or another receptor recognizing a nucleic acidmolecule of the Inositol hexakisphosphate kinase or RYK kinase genefamily or a polypeptide encoded thereby for identifying substancescapable of interacting with an Inositol hexakisphosphate kinase or RYKkinase homologous polypeptide.
 16. A non-human transgenic animalexhibiting a modified expression of an Inositol hexakisphosphate kinaseor RYK kinase homologous polypeptide.
 17. The animal of claim 1 6,wherein the expression of the Inositol hexakisphosphate kinase or RYKkinase homologous polypeptide is increased and/or reduced.
 18. Arecombinant host cell exhibiting a modified expression of an Inositolhexakisphosphate kinase or RYK kinase homologous polypeptide.
 19. Thecell of claim 18 which is a human cell.
 20. A method of identifying a(poly)peptide involved in the regulation of energy homeostasis and/ormetabolism of triglycerides in a mammal comprising the steps of (a)contacting a collection of (poly)peptides with an Inositolhexakisphosphate kinase or RYK kinase homologous polypeptide or afragment thereof under conditions that allow binding of said(poly)peptides; (b) removing (poly)peptides which do not bind and (c)identifying (poly)peptides that bind to said Inositol hexakisphosphatekinase or RYK kinase homologous polypeptide.
 21. A method of screeningfor an agent which modulates the interaction of an Inositolhexakisphosphate kinase or RYK kinase homologous polypeptide with abinding target/agent, comprising the steps of (a) incubating a mixturecomprising (aa) an Inositol hexakisphosphate kinase or RYK kinasehomologous polypeptide, or a fragment thereof; (ab) a bindingtarget/agent of said Inositol hexakisphosphate kinase or RYK kinasehomologous polypeptide or fragment thereof; and (ac) a candidate agentunder conditions whereby said Inositol hexakisphosphate kinase or RYKkinase polypeptide or fragment thereof specifically-binds to saidbinding target/agent at a reference affinity; (b) detecting the bindingaffinity of said Inositol hexakisphosphate kinase or RYK kinasepolypeptide or fragment thereof to said binding target to determine an(candidate) agent-biased affinity; and (c) determining a differencebetween (candidate) agent-biased affinity and the reference affinity.22. A method of screening for an agent which modulates the activity ofan Inositol hexakisphosphate kinase or RYK kinase homologous polypeptidecomprising the steps of (a) incubating a mixture comprising (aa) anInositol hexakisphosphate kinase or RYK kinase homologous polypeptide,or a fragment thereof, and (ab) a candidate agent under conditionswhereby said Inositol hexakisphosphate kinase or RYK kinase polypeptideor fragment thereof has a reference activity, (b) detecting the activityof said Inositol hexakisphosphate kinase or RYK kinase polypeptide orfragment thereof to determine an (candidate) agent-biased activity and(c) determining a difference between (candidate) agent-biased activityand the reference activity.
 23. A method of producing a compositioncomprising the (poly)peptide identified by the method of claim 20 or theagent identified by the method of claim 21 or 22 with a pharmaceuticallyacceptable carrier, diluent and/or adjuvant.
 24. The method of claim 23wherein said composition is a pharmaceutical composition for preventing,alleviating or treating of diseases and disorders, including metabolicdiseases such as obesity and other body-weight regulation disorders aswell as related disorders such as metabolic syndrome, eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones and otherdiseases and disorders.
 25. Use of a (poly)peptide as identified by themethod of claim 20 or of an agent as identified by the method of claim21 or 22 for the preparation of a pharmaceutical composition for thetreatment, alleviation and/or prevention of of diseases and disorders,including metabolic diseases such as obesity and other body-weightregulation disorders as well as related disorders such as metabolicsyndrome, eating disorder, cachexia, diabetes mellitus, hypertension,coronary heart disease, hypercholesterolemia, dyslipidemia,osteoarthritis, gallstones and other diseases and disorders.
 26. Use ofa nucleic acid molecule of the Inositol hexakisphosphate kinase or RYKkinase family or of a fragment thereof for the preparation of anon-human animal which over- or under-expresses the Inositolhexakisphosphate kinase or RYK kinase gene product.
 27. Kit comprisingat least one of (a) an Inositol hexakisphosphate kinase or RYK kinasenucleic acid molecule or a fragment thereof; (b) a vector comprising thenucleic acid of (a); (c) a host cell comprising the nucleic acid of (a)or the vector of (b); (d) a polypeptide encoded by the nucleic acid of(a); (e) a fusion polypeptide encoded by the nucleic acid of (a); (f) anantibody, an aptamer or another receptor against the nucleic acid of (a)or the polypeptide of (d) or (e) and (g) an anti-sense oligonucleotideof the nucleic acid of (a).
 28. Nucleic acid molecule comprising (a) thenucleotide sequence as shown in SEQ ID NO: 7 and/or a sequencecomplementary thereto, or (b) a sequence which is degenerated withrespect to the nucleic acid molecule of (a) or (c) a fragment of thenucleic acid molecule of (a) or (b) having a length of at least 20nucleotides.
 29. A polypeptide or peptide encoded by the nucleic acidmolecule of claim 28.